This invention relates to nucleic acid and amino acid sequences of cell surface glycoproteins and to the use of these sequences in the diagnosis, treatment, and prevention of hematologic, karyotypic, and neuronal disorders.
Proteins expressed on the cell surface function as stage-specific markers of cell differentiation and as antigenic determinants for immunological identification of distinct cell types. In the context of tissue construction and intercellular communication, cell surface proteins play critical roles in cell-cell recognition and adhesion, cell motility, and signal transduction. Cell surface proteins are anchored to the plasma membrane by one or more membrane-spanning domains or by covalent attachment to lipophilic membrane-embedded molecules such as glycosylphosphatidylinositol. Most cell surface proteins are synthesized as immature, inactive precursors which require post-translational modifications for activity. Such modifications include proteolytic processing, glycosylation, oligomerization, and disulfide bond formation. Proteins destined for the cell surface generally contain N-terminal signal peptides which are cleaved from the mature protein.
Blood group antigens are immunologically defined marker proteins found on the surface of red blood cells. These markers provide a means for classification of blood group systems. For example, the most commonly known blood group system is defined by the A, B, and O antigens. The particular antigen(s) expressed by a given individual is determined by the allele, or variant, of the ABO gene that the individual has inherited. The blood type of the individual is therefore defined by the serologically expressed antigen(s). The implications of the ABO system on blood transfusion practice are well known. In addition, the A and B antigens have potential application in the diagnosis of blood disorders such as leukemia, thalassemia, and anemia which weaken the expression of these antigens. (Reviewed in Reid, M. E. and Lomas-Francis, C. (1997) The Blood Group Antigen Facts Book, Academic Press, San Diego, Calif., pp. 19-26.)
Although the ABO system is well known for its clinical relevance, other blood group systems exist. One such blood group system is the XG system which is defined by a single antigen, Xga. Xga is encoded by one of two allelic forms of the XG gene. The other XG allele fails to encode a detectable gene product. (Reviewed in Reid and Lomas-Francis, supra, pp. 251-254.) The XG gene is situated on the X chromosome at the boundary between the pseudoautosomal, or X-specific, region and the region which has homology to Y chromosome sequences. (Ellis, N. A. et al. (1994) Nat. Genet. 6:394-399.) The latter region is particularly important for recombination between the X and Y chromosomes during male meiosis. Lack of recombination results in the failure of X and Y to segregate and ultimately leads to the generation of male progeny with an XXY karyotype. XXY individuals suffer from Klinefelter syndrome, a complex developmental disorder characterized by infertility, gynecomastia and other manifestations of feminization, increased height, obesity, mental deficiency, thyroid abnormalities, diabetes, pulmonary disease, and increased risk of breast cancer.
The XG gene has been cloned and sequenced (Ellis, supra, and Ellis, N. A. et al. (1994) Nat. Genet. 8:285-289). XG RNA is detectable at low levels in fibroblasts and in some bone marrow preparations. XG cDNA predicts a proline- and glycine-rich protein of 180 amino acids with an N-terminal signal peptide. A transmembrane domain from amino acid 88 through 116 separates the N-terminal extracellular domain from the C-terminal intracellular domain. The XG protein may play a role in cell adhesion.
The surface of white blood cells, like that of red blood cells, is populated with characteristic glycoproteins. For example, the plasma cell glycoprotein-1 (PC-1) is expressed on the surface of plasma cells, which are terminally differentiated, antibody-secreting B-lymphocytes. PC-1 is also expressed in nonlymphoid tissue such as kidney, chondrocytes, epididymis, and hepatocytes. PC-1 was initially isolated from murine plasma cells as a homodimer with subunits of 115 kilodaltons each (van Driel, I. R. et al. (1985) Proc. Natl. Acad. Sci. USA 82:8619-8623). Biochemical and immunological analyses have suggested that murine PC-1 (mPC-1) is expressed in neuroblastomas. However, molecular analyses failed to confirm this observation, suggesting that neuroblastoma tissue contains a glycoprotein having biochemical similarity to or immunological cross-reactivity with mPC-1. mPC-1 cDNA encodes a predicted protein of 871 amino acids with a short N-terminal cytoplasmic domain, a single transmembrane domain, and a large C-terminal extracellular domain of 826 amino acids. Human PC-1 (hPC-1) is 873 amino acids in length and 80% identical to the mouse protein (Buckley, M. F. et al. (1990) J. Biol. Chem. 265:17506-17511). hPC-1 possesses the same overall domain structure as mPC-1. In addition, a soluble form of mPC-1 has been found in serum and other extracellular fluids (Belli, S. I. et al. (1993) Eur. J. Biochem. 217:421-428). This soluble form of mPC-1 likely results from proteolytic cleavage which frees most of the extracellular domain from the transmembrane domain.
The extracellular domains of both hPC-1 and mPC-1 have nucleotide phosphodiesterase (pyrophosphatase) activity (Funakoshi, I. et al. (1992) Arch. Biochem. Biophys. 295:180-187; Rebbe, N. F. et al. (1991) Proc. Natl. Acad. Sci. USA 88:5192-5196). In hPC-1, the enzymatic active site for this activity likely occurs within the region from amino acids 166 through 225. Phosphodiesterase activity is associated with the hydrolytic removal of nucleotide subunits from oligonucleotides. Although the precise physiological roles of hPC-1 and mPC-1 are not clear, increased hPC-1 phosphodiesterase activity has been correlated with insulin resistance in patients with noninsulin-dependent diabetes mellitus, with abnormalities of bone mineralization and calcification, and with defects in renal tubule function. In addition, it appears that hPC-1 and mPC-1 are members of a multigene family of transmembrane phosphodiesterases with extracellular active sites. These enzymes may play a role in regulating the concentration of pharmacologically active extracellular compounds such as adenosine or other nucleotide derivatives in a variety of tissues and cell types. (Reviewed in Goding, J. W. et al. (1998) Immunol. Rev. 161:11-26.)
The discovery of new cell surface glycoproteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of hematologic, karyotypic, and neuronal disorders.
The invention features substantially purified polypeptides, cell surface glycoproteins, referred to collectively as xe2x80x9cCSGPxe2x80x9d and individually as xe2x80x9cCSGP-1xe2x80x9d and xe2x80x9cCSGP-2. xe2x80x9d In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide-, and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof. The invention also provides a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of CSGP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of CSGP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof.